Protein aggregates are implicated in a host of diseases including Alzheimer's, Parkinson's, Type II diabetes, heart and prion diseases, and are also being increasingly found to be of functional significance. While amyloid fibrils were previously believed to be the toxic species, more recent data suggest a major disease-associated role for smaller oligomers that are intermediates in fibril formation. Because amyloid formation occurs on a high-dimensionality and complex reaction landscape, structural features of intermediates in the reaction are often hidden in standard ensemble studies. Single molecule studies hold great promise for uncovering the full distributions of structural properties for the intricate mixture of oligomeric intermediates in such reactions. However, because single molecule studies are typically carried out at low concentrations (nM and below) and under equilibrium conditions, they are often not compatible with the high-concentrations, nonequilibrium conditions and unstable oligomers encountered during amyloid formation. In this project, we will focus on developing a novel rapid-dilution dual-color coincidence method for studying oligomerization-number distributions of such weak oligomeric intermediates, by using the combined strengths of single molecule experiments and microfluidics. The key underlying idea behind the methodology is that dilution from the aggregation reaction will be achieved very rapidly, followed immediately by single molecule detection, thus allowing single molecule structural studies of the weak oligomers prior to dissociation. The method will be developed using a model DNA system, which will provide a set of oligomers with well-defined and tunable properties such as oligomerization state and stability, essential in allowing us to explore the strengths of the method, as well as to develop experimental protocols and analysis routines for optimally extracting oligomerization information from single molecule fluorescence bursts. The developed coincidence method will then be applied to study oligomer distributions during the early phases of protein aggregation. The ultimate goal of the method in this context is to simultaneously provide information about protein oligomerization (via coincidence measurements) and conformational distributions (e.g., using FRET) as a function of time during the early stages of amyloid formation. The developed method will also be very generally useful for the detailed study of structure and dynamics of weak complexes widely prevalent in the cell. PUBLIC HEALTH RELEVANCE This project aims to develop and apply a novel single-molecule fluorescence methodology to probe distributions of unstable oligomeric intermediates in amyloid formation. This methodology is anticipated to provide a detailed mapping of the structural properties of such unstable oligomers, valuable for a basic understanding of their cellular functions and disorders, and during the design of therapeutic strategies against several amyloid diseases.